Proteolytic activity driven by urokinase plasminogen activator (uPA) is commonly recognized as a critical factor in metastasis. Reduction of proteolytic activity has been proposed as a cancer treatment option; uPA inhibitors could be utilized as a cancer treatment if used prior to the onset of the metastatic process. Moreover, uPA inhibitors have been recently shown to reduce tumor growth as well. We have found that uPA inhibitors reduce angiogenesis (uPA is overexpressed on tip of capillary vessels) and thereby reduce tumor size. The ultimate goal of this proposal is to identify novel inhibitors of uPA suitable for cancer treatment. In this study we want make use of plasminogen activator inhibitor (PAI-1), which will be genetically engineered to introduce novel and desired properties to this protein. These properties include: long half-life (wild PAI-1 converts into its latent form in approximately 2h), lack of vitronectin binding capability (PAI-1 binds to this protein and this property could increase motility of cancer cells), and uPA specificity (PAI-1 is a non-specific inhibitor of both uPA and tPA). The urokinase is involved in pericellular malignant proteolysis, while tPA mainly mediates physiologically needed intravascular thrombolysis and that function ought to be preserved. Lastly, these altered forms of PAI-1 will be combined and tested for anti-uPA and anti-angiogenic activity. The metastatic process and angiogenesis are driven by the urokinase plasminogen system and matrix metallo proteinases (MMPs). In some clinical studies of advanced cancers, inhibition of MMPs showed promise, while in other studies they did not. The efficacy of anti-angiogenic agents would probably be best in remission after traditional chemotherapy, or in combination with cytotoxic agents and possibly with combinations of urokinase and MMPs inhibitors. In addition, the best results can perhaps be gained in adjuvant therapy or in early stages of cancer, when the tumor burden is minimal. Our future efforts are directed toward these goals.